IMMORTALIZED CELLS FOR CYSTIC FIBROSIS RESEARCH

已经发现了疾病特有的离子转运异常 在CF呼吸道和其他受影响的器官中导致了这一假说 上皮性离子细胞的变化表现出类似的 异常，证明这些生理异常 是上皮细胞固有的，而不是继发性的 炎症或循环介质。主服务器的可用性 Cf上皮细胞培养使分子研究成为可能。 对观察到的变化负责的机制，但很少有组织 样品是可用的，细胞的存活时间很短。 因此，CF研究继续受到以下方面的限制 充足的研究材料。这个项目将 增殖性CF型呼吸道上皮细胞系的建立 能力和良好的形态和表型特征 属性。我们将利用猿猴的永生基因 病毒40用于这些研究，基于其产生人的记录 具有分化特性的上皮细胞系。这个 选定要使用的构件的原点有缺陷 复制，并包括对温度敏感的突变体 SV40T区。这些基因将通过感染 腺病毒/SV40混合型病毒，逆转录病毒感染，以及 电穿孔和DEAE/葡聚糖共转化载体。 克隆将通过对抗生素G418和 通过延长生存时间。细胞将在塑料培养皿中培养， 可渗透的胶原基质支架，并在异种气管中 嫁接。细胞将在连续传代时进行超低温保存 提供用于运送至囊性纤维化的细胞系库存 调查人员。长时间生长的细胞系将是 具有位相对比度、光、透射电子显微镜和冷冻断裂的特征 形态研究。SV40T的上皮性质及表达 抗原将用单抗检测来确认。这个 将评估生理特性(特别是离子传输) 使用多种技术，包括跨上皮生物电 研究，细胞内和离子特异性微电极，贴片 夹具，以及潜在的敏感荧光染料。这些 研究可能会产生6到20个危机前或危机后的细胞 取自CF和正常呼吸道上皮细胞的细胞系。这些细胞的等量 来自特定通道编号的行将提供给 在拨款过程中与CF研究人员合作。

Disease specific ion transport abnormalities have been identified in CF airways and other affected organs leading to the hypothesis that alterations in epithelial ion cells demonstrate similar abnormalities, establishing that these physiologic abnormalities are intrinsic to the epithelial cells and not secondary effects of inflammation or circulating mediators. The availability of primary CF epithelial cultures has permitted investigation of the molecular mechanisms responsible for the observed changes, but few tissues samples are available and the cells have a short survival. Therefore, CF research continues to be limited by availability of adequate quantities of research materials. This project will develop CF airway epithelial cell lines with increased growth capability and well-characterized morphologic and phenotypic properties. We will utilize the immortalizing genes of Simian Virus 40 for these studies, based on its record of producing human epithelial cell lines with differentiated properties. The constructs selected for use are deficient in the origin of replication, and include a temperature sensitive mutant of the SV40T region. These genes will be introduced by infection with hybrid adeno/SV40 viruses, infection with retroviruses, and by transfection of plasmids with electroporation and DEAE/dextran. Clones will be selected by resistance to the antibiotic, G418, and by prolonged survival. Cells will be cultured on plastic dishes, permeable collagen matrix supports, and in heterologous trachea grafts. Cells will be cryopreserved at sequential passages to provide stocks of cells lines for shipping to cystic fibrosis investigators. Cell lines with prolonged growth will be characterized by phase contrast, light, TEM, and freeze-fracture morphologic studies. The epithelial nature and expression of SV40T antigen will be confirmed with monoclonal antibody assays. The physiologic (especially ion transport) properties will be assessed with multiple techniques, including transepithelial bioelectric studies, intracellular and ion specific microelectrodes, patch clamp, and with potential sensitive fluorescent dyes. These studies are likely to produce 6 to 20 pre- or post-crisis cell lines from CF and normal airway epithelia. Aliquots of these cells lines from specific passage numbers will be made available to collaborating CF researchers during the course of the grant.